Systems and methods for analyzing an extracted sample

ABSTRACT

The invention generally relates to systems for analyzing a sample and methods of use thereof. In certain aspects, the invention provides systems that include an ionization probe and a mass analyzer. The probe includes a hollow body that has a distal tip. The probe also includes a substrate that is at least partially disposed within the body and positioned prior to the distal tip so that sample extracted from the substrate flows into the body prior to exiting the distal tip. The probe also includes an electrode that operably interacts with sample extracted from the substrate.

RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationof PCT/US14/11000, filed Jan. 10, 2014, which claims the benefit of andpriority to each of U.S. provisional patent application Ser. No.61/779,673, filed Mar. 13, 2013, and U.S. provisional patent applicationSer. No. 61/759,247, filed Jan. 31, 2013, the content of each of whichis incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under GM103454 awardedby the National Institutes of Health and CHE0847205 awarded by theNational Science Foundation. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention generally relates to systems and methods for analyzing anextracted sample.

BACKGROUND

Chemical analysis using mass spectrometry traditionally involves sampleextraction and chromatographic separation prior to mass analysis. Forexample, biofluids (e.g., complex mixtures such as blood, saliva, orurine) are routinely separated using chromatography before a massspectrometry measurement in order to minimize suppression effects onanalyte ionization and to pre-concentrate the analytes. Recently,systems and methods have been developed that allow for samplepreparation and pre-treatment to be combined with the ionization process(See Ouyang et al., WO 2010/127059, the content of which is incorporatedby reference herein in its entirety).

Those systems and methods use wetted porous material, named paper sprayionization, for direct, qualitative and quantitative analysis of complexbiofluids. Analyte transport is achieved by wicking in a porous materialwith a macroscopically sharp point and a high electric field is used toperform ionization and chemical analysis of compounds present inbiological samples. Pneumatic assistance is not required to transportthe analyte; rather, a voltage is simply applied to the wet paper thatis held in front of a mass spectrometer.

SUMMARY

The invention recognizes that a short coming of paper spray is that itgenerates short and unstable spray due to a fast drying of solvent onpaper when operated with mass spectrometers using curtain gases.Additionally, paper spray has low sensitivity with miniature massspectrometers due to relatively poorer desolvation. The invention solvesthose problems by providing a housing for the substrate that includes aspray tip.

The invention operates similar to paper spray in that sample is appliedto a substrate. However, unlike paper spray, the sample is not directlyionized from the substrate. Rather, solvent is applied within thehousing to interact with the substrate and extract sample analytes fromthe substrate. The sample analytes in the extraction solvent remain inan aqueous phase until application of a voltage to within the housing.At that time the analytes in the extraction solvent are expelled fromthe distal tip of the housing, thereby generating ions of the analytes.Probes of the invention are particularly suitable for use withnebulizing gas and have improved desolvation over paper spray.

In certain aspects, the invention provides systems that include anionization probe and a mass analyzer. The probe includes a hollow bodythat has a distal tip. The probe also includes a substrate that is atleast partially disposed within the body and positioned prior to thedistal tip so that sample extracted from the substrate flows into thebody prior to exiting the distal tip. In certain embodiments, thesubstrate is completely within the body. The probe also includes anelectrode that operably interacts with sample extracted from thesubstrate. The electrode may be outside the body, fully disposed withinthe body, or only partially disposed within the body. The hollow bodymay be made of any material, and an exemplary material is glass. Thehollow body may include a port for receiving a solvent. Alternatively,solvent is introduced to the substrate and enters the body by flowingthrough the substrate.

The substrate can be porous or non-porous material. In certainembodiments, the substrate is a porous material. Any porous material,such as polydimethylsiloxane (PDMS) membranes, filter paper, cellulosebased products, cotton, gels, plant tissue (e.g., a leaf or a seed)etc., may be used as the substrate. The mass analyzer may be for a massspectrometer or a miniature mass spectrometer. Exemplary mass analyzersinclude a quadrupole ion trap, a rectalinear ion trap, a cylindrical iontrap, an ion cyclotron resonance trap, or an orbitrap.

In certain embodiments, the system further includes a source ofnebulizing gas. The source of nebulizing gas may be configured toprovide pulses of gas. Alternatively, the source of nebulizing gas maybe configured to provide a continuous flow of gas.

Another aspect of the invention provides methods for analyzing a sample.The methods involve introducing a solvent to a sample on a substratethat is at least partially disposed within a hollow body such that thesolvent interacts with the substrate to extract to the sample from thesubstrate, applying a voltage to the extracted sample in the solvent sothat the sample is expelled from a distal tip of the body, therebygenerating ions of an analyte in the sample, and analyzing the ions. Thesubstrate may be completely disposed within the body or only partiallydisposed within the body. In certain embodiments, a nebulizing gas isalso applied to the extracted sample. The sample may be introduced tothe substrate prior to the substrate being at least partially insertedinto the hollow body. Alternatively, the sample may be introduced to thesubstrate after the substrate has been partially inserted into thehollow body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of an extraction spray ion source for MSanalysis. FIG. 1B is a schematic of the extraction spray ionizationprocess, with two proposed steps: extraction and spray ionization. FIG.1C is an extraction spray-MS/MS spectrum for dried blood analysis using10 μL methanol as spray solvent, 0.2 μL blood containing 10 ng/mLamitriptyline. FIG. 1D is a set of photographs of loaded samples beforeand after extraction spray process with different solvents (puremethanol, methanol/water 50/50 and pure water).

FIGS. 2A-B are ion chronograms for the product ion m/z 283 of sunitinib,prepared by 0.2 μL, 200 ng/mL sunitinib in blood samples, using massspectrometers with different API. FIG. 2A: TSQ with a heated capillaryAPI. FIG. 2B: Sciex QTRAP4000 with a curtain gas API. The ionchronograms by extraction spray (top lines) and paper sprays (bottomlines) were compared using both instruments. Mass spectrometers were seton single reaction monitoring (SRM) mode, and 10 μL of methanol was usedas extraction solvent. FIG. 2C is a calibration curve of amitriptyline,monitoring the intensity of the fragment ion m/z 233 using 10 μLmethanol as solvent and 0.2 μL DBSs containing amitriptyline and[D6]amitriptyline as standard.

FIGS. 3A-F are mass spectra for chemicals in different matrices andcorresponding tandem mass spectra using a Sciex QTRAP 4000. Spectra wereobtained in the positive ion mode with a spray voltage 2 kV: (FIG. 3A)nicotine in dried blood spots (DBSs), (FIG. 3B) methamphetamine in DBSs,(FIG. 3C) methamphetamine in urine, (FIG. 3D) clenbuterol in porkhommogenate, (FIG. 3E) atrazine in river water, and (FIG. 3F)thiabendazole in orange homogenate.

FIG. 4 is a graph showing quantitation of therapeutic drugs in bloodsample using Mini 12 mass spectrometer with extraction spray.Calibration curve for amitriptyline in bovine blood withamitriptyline-d6 (100 ng/ml) as internal standard. SRM m/z 278 to 233and m/z 284 to 233 was used for analyte and internal standard,respectively. Sample Whatman Grad 1 chromatography paper, 0.18 mmthickness, 8 mm long, 0.8 mm wide. Dried blood spot prepared with 2 μLblood sample. 7 μL methanol used for extraction spray. 1800 V appliedfor spray.

FIG. 5 is a schematic showing a discontinuous atmospheric pressureinterface coupled in a miniature mass spectrometer with rectilinear iontrap.

FIG. 6 is a schematic showing an extraction spray probe in which thesubstrate is only partially disposed within the body (spray tip). TheDAPI is an optional component of the system and the substrate shapeshown is an exemplary shape with exemplary dimensions.

DETAILED DESCRIPTION

The invention provides extraction spray ionization for direct analysisof raw samples with complex matrices. In certain embodiments, systems ofthe invention include an ionization probe. An exemplary probe is shownin FIG. 1A. The probe includes a hollow body that has a distal tip. Anexemplary hollow body is one similar to that used for nanoESI. Exemplarynano spray tips and methods of preparing such tips are described forexample in Wilm et al. (Anal. Chem. 2004, 76, 1165-1174), the content ofwhich is incorporated by reference herein in its entirety. A substrateis at least partially disposed within the body and positioned prior tothe distal tip so that sample extracted from the substrate flows intothe body prior to exiting the distal tip. In certain embodiments, suchas shown in FIG. 1A, the substrate is completely within the body. Inother embodiments, such as shown in FIG. 6, the substrate is onlypartially disposed within the body (spray tip). The hollow body mayinclude a port for receiving a solvent (FIG. 1A). Alternatively, solventmay be introduced to the substrate and enters the body by flowingthrough the substrate (FIG. 6). The probe also includes an electrodethat operably interacts with sample extracted from the substrate. Theelectrode may be outside the body (FIG. 6), fully disposed within thebody, or only partially disposed within the body (FIG. 1A). The probe isoperably coupled to a mass spectrometer, such that ions produced by theprobe enter the mass spectrometer. The invention combines a fastextraction with an ionization process, such as nanospray, which allowsdirect analysis of raw samples and a much improved spray ionization toprovide a good sensitivity to ambient analysis using a wide variety ofmass spectrometers.

Extraction spray includes a fast extraction of the analytes from sampleon a substrate and a subsequent spray of the extraction solution using aspray tip. Based on the extraction-ionization model proposed, extractionspray can be viewed as a two-step process, as demonstrated in FIG. 1B.At the extraction step, extraction solvent rapidly extracts analytematrices from a dried sample, such as dried blood spots or dried tissuehomogenates, which were deposited on a sample substrate within a nanoESItube. Similar to the paper spray process, the differences on extractionefficiencies of solvents to analytes as well as adsorbing powers ofsamples to substrates are expected to have significant impact on thisstep. Followed by the fast extraction, the extractants entrained insolvent are sprayed and ionized. In the exemplary embodiment shown inFIGS. 1A-B, that process is a nanoESI-like process. The charged dropletsgenerated by extraction spray have a much smaller size as compared todroplets produced by paper spray. Without being limited by anyparticular theory or mechanism of action, it is believed that thesmaller droplet size produced by systems of the invention is due to itssimilar droplet generation as nanoESI, and a more efficient gas phasecharged droplet desolvation process which occurs prior to the spraydroplets entrance into a mass analyzer. Thus, this simple approach hasthe potential to elevate the performance of miniature mass spectrometersin which desolvation strategies are seldom applied as a compromise toportability.

Extraction spray has both good sensitivity, similar to that of nanoESI,and high matrix tolerance, similar to that of paper spray. FIG. 1C showsthe extraction spray-MS result for the analysis of dried blood spots(DBSs) on paper substrates with 0.2 μL whole blood samples containing 10ng/mL amitryptline. With only 0.2 μL sample, ultralow concentration ofamitriptyline (10 ng/mL) was able to be detected from the DBS. 10 μL ofMethanol and water mixed with different volume ratio were used assolvents for the test. Photographs of the sampling strips were takenbefore and after the DBS analysis using methanol/water (100/0, 50/50 and0/100, v/v ratio) as extraction solvents (FIG. 1D). The increase of theaqueous component in the solvent system was found to extract morematerials from the DBSs into the solvent phase, which was beneficial tothe blood analysis using extraction spray-MS.

The signal stabilities and durations of extraction spray and paper spraywere compared using mass spectrometers of different APIs: a heatedcapillary API (TSQ) and a curtain gas API (Sciex QTRAP4000). Forextraction spray, 0.2 μL samples, 200 ng/mL sunitinib in blood, werepreloaded and dried on paper strips before insertions into nanoESItubes. Extraction solvent, 10 μL methanol, was consequently addedthrough the end of the tubes, and constant sprays were formed with theassistance of a spray voltage of 2 kV. Paper spray operations similar toprevious studies were used: the same amount of samples, 0.2 μL sunitinibin bovine blood, were spotted and dried on the centers of papertriangles, and elution solvent of 10 μL methanol was applied forgenerating a stable spray. About 3.5 k DC voltage was used to facilitatepaper spray. The chronogram for product ion m/z 283 were recorded usingsingle reaction monitoring mode (SRM) on both TSQ and QTRAP4000 massspectrometers. With a heated capillary API, paper spray was able togenerate an intensive chronogram with a bimodal pattern: product ion ofgood abundance was generated at the beginning followed by a decrease insignal intensity, and the abundance of product ion increased to an evenhigher level before the final signal decay as the expiration of elutionsolvent happened around 1.0 min (FIG. 2A, bottom signal).

In contrast, extraction spray demonstrated a stable signal with a muchlonger signal duration (>9.0 min) but a little lower signal abundance(FIG. 2A, top signal). More significant differences of the ionchronograms between the two methods were observed when using a SciexQTRAP4000 with a curtain gas API. Stable signals with long duration (>9min) were generated by extraction spray (FIG. 2B, top signal) and abimodal ion chronogram with good signal abundance of less than 20.0 secwas obtained in paper spray (FIG. 2B, bottom signal). In general, thesignal of extraction spray was able to be maintained for longer than 30min. The signal intensities of paper spray were slightly higher thanextraction spray in both cases, but of significantly shorter duration.The spray current of both methods were measured respectively. Higherspray but dynamic spray current was generated during paper spray process(0.17-0.77 μA), while the spray current stayed constant, 0.28 μA, inextraction spray. Considering the absence of flow dynamics control inpaper spray, the observations of dynamic signal produced in paper spraywere believed to be caused by continuous reduction of the solvent amounton the paper substrate and the difference in the desolvation of chargeddroplets which were derived from Taylor cone jets. In other words, evenhighly charged droplets were formed during papers spray at reducing flowrates. Only a portion of the droplets having a smaller size were able tobe completely desolvated within the APIs to form detectable ions. Thereduction of signal duration in paper spray with the curtain gas API wasowed to a faster solvent vaporization on the paper substrate facilitatedby curtain gas flow. The signal duration in extraction spray was able tobe maintained because of the protection of the solvent in the glassspray tube from the gas flow. Paper spray has demonstrated a strongquantitation capability using mass spectrometer of heated capillary APIbecause the signal variations are able to be reduced by integratingsignals over a longer acquisition time (typically >30 sec). However,limited by shorter signal duration, coupling paper spray-MS with acurtain gas API is a challenge. Systems and methods of the invention(i.e., extraction spray) solve that problem as illustrated by the datashown in FIGS. 2A-B.

An assessment of the quantitation potential of extraction spray wasconducted by using a therapeutic drug, amitriptyline m/z 277, preparedin whole bovine blood samples. The quantitation of amitriptyline wasobtained by using the intensity ratios of a product ion m/z 233 ofamitriptyline to the corresponding fragment ion produced from[D6]amitriptyline which was added to amitriptyline samples as internalstandard (FIG. 2C). The relative response is across a linear range 7-700ng/mL with R²=0.9991 covering the therapeutic range of amitriptyline(80-250 ng/mL). The relative standard deviations are less than 5% at alldata points. Similar or better performances could be expected forquantitation of other small molecules from raw samples. In certainembodiments, the housing can include a coating of an internal standard,which allows for ultrafast MS analysis of complex sample.

The versatility of extraction spray was characterized using a variety ofchemicals which were prepared in complex matrices such as dried bloodspots (DBSs) and tissue homogenates (FIGS. 3A-C). All the mass spectraand MS/MS spectra were acquired using extraction spray with 0.2 μLsamples loaded on sample substrates and dried in air. The solventcondition was optimized by comparing the intensity of product ion m/z 91of methamphetamine 200 ng/mL in DBSs, and 10 μL of methanol wasdetermined as the extraction solvent based on the comparison. Similar topaper spray, all the chemicals demonstrated pseudo-molecular ion as theform [M+H]⁺. In the analysis of psychoactive drugs, the mass spectra fornicotine in DBSs and methamphetamine in urine and DBSs were acquired(FIGS. 3A-C). Both MS and MS/MS spectra of methamphetamine in urine wereobserved with good S/N ratio. Although the drug peaks formethamphetamine and nicotine were overwhelmed by matrices in DBSsanalysis, MS/MS spectrum with good S/N was able to be obtained at theconcentration level of 200 ng/mL. In the analysis of foodcontaminations, 10 ng/mL clenbuterol in pork homogenate, product ions ofgood abundances could be observed in MS/MS spectra using 0.2 μL samplesat concentration level of 10 ng/mL (FIG. 3D). For agriculture chemicalscreening, the ion signals of atrazine and thiabendazol of good S/Nratio in MS and MS/MS spectra were able to be observed at the ultralowconcentration: 50 ng/mL and 1 ng/mL respectively (FIGS. 3E-F). Thelimits of detection (LODs) of chemicals in raw samples were determined(Table 1).

TABLE 1 Limits of detection (LODs) of chemicals in various matricesusing extraction spray method. LOD Chemicals Category Matrix (ng/mL)Melamine Contaminant Milk 1 Clenbuterol Contaminant Pork homogenate 0.5Atrazine Herbicide River water 0.1 Thiabendazole Fungicide Orangehomogenate 0.1 Methamphetamine Psychoactive drug Blood 0.1 NicotinePsychoactive drug Blood 1 Imatinib Therapeutic drug Blood 1 VerapamilTherapeutic drug Blood 0.5 Sunitinib Therapeutic drug Blood 1Good sensitivity and high matrix tolerance could be achieved bycombining the extraction and the spray ionization. As discussed above,the new ion source can be used for analysis of a wide variety ofchemical species, including psychoactive/therapeutic drugs, foodcontaminations and agricultural chemicals.

Sensitive and reliable result were achieved using ambient massspectrometry with a combination of fast extraction and spray ionization(i.e., extraction spray). Durable and stable signals were produced byextraction spray when coupled with mass spectrometers of curtain gas APIand heated capillary API. Linear response of 7-700 ng/mL was achieved inthe quantitation of amitriptyline in whole blood samples. The detectionsof a variety of low concentration chemicals in different matricesdemonstrates broad applications of this hybrid method.

Probes of the invention can be coupled to any type of mass analyzers andatmospheric pressure interfaces known in the art. Exemplary massanalyzers are a quadrupole ion trap, a rectalinear ion trap, acylindrical ion trap, an ion cyclotron resonance trap, or an orbitrap.Probes of the invention can be coupled to interfaces and mass analyzersthat utilize curtain gas. Such an exemplary system is an API (SciexQTRAP4000). Alternatively, probes of the invention can be coupled tointerfaces and mass analyzers that do not utilize curtain gas.

The mass analyzer may be for a bench-top or lab-scale mass spectrometeror a miniature mass spectrometer. An exemplary miniature massspectrometer is described, for example in Gao et al. (Z. Anal. Chem.2008, 80, 7198-7205), the content of which is incorporated by referenceherein in its entirety. In comparison with the pumping system used forlab-scale instruments with thousands watts of power, miniature massspectrometers generally have smaller pumping systems, such as a 18 Wpumping system with only a 5 L/min (0.3 m3/hr) diaphragm pump and a 11L/s turbo pump for the system described in Gao et al. Other exemplaryminiature mass spectrometers are described for example in Gao et al.(Anal. Chem., 2006, 80:7198-7205, 2008), Hou et al. (Anal. Chem.,83:1857-1861, 2011), and Sokol et al. (Int. J. Mass Spectrom., 2011,306, 187-195), the content of each of which is incorporated herein byreference in its entirety.

Substrates and Solvents

Exemplary substrates are described, for example in Ouyang et al. (U.S.patent application number 2012/0119079) and Ouyang et al. (U.S. patentapplication Ser. No. 14/119,548), the content of each of which isincorporated by reference herein in its entirety. In certainembodiments, the porous material is any cellulose-based material. Inother embodiments, the porous material is a non-metallic porousmaterial, such as cotton, linen, wool, synthetic textiles, or glassmicrofiber filter paper made from glass microfiber. In certainembodiments, the substrate is plant tissue, such as a leaf, skin or barkof a plant, fruit or vegetable, pulp of a plant, fruit or vegetable, ora seed. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (8 μm), Grade 595(4-7 μm), and Grade 6 (3 μm), Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultra-filtration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

In other embodiments, the porous material is treated to producemicrochannels in the porous material or to enhance the properties of thematerial for use in a probe of the invention. For example, paper mayundergo a patterned silanization process to produce microchannels orstructures on the paper. Such processes involve, for example, exposingthe surface of the paper totridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to result insilanization of the paper. In other embodiments, a soft lithographyprocess is used to produce microchannels in the porous material or toenhance the properties of the material for use as a probe of theinvention. In other embodiments, hydrophobic trapping regions arecreated in the paper to pre-concentrate less hydrophilic compounds.Hydrophobic regions may be patterned onto paper by usingphotolithography, printing methods or plasma treatment to definehydrophilic channels with lateral features of 200-1000 μm. See Martinezet al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al.(Proc. Natl Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal.Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80,3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li et al.(Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. Liquid samples loadedonto such a paper-based device can travel along the hydrophilic channelsdriven by capillary action.

Another application of the modified surface is to separate orconcentrate compounds according to their different affinities with thesurface and with the solution. Some compounds are preferably absorbed onthe surface while other chemicals in the matrix prefer to stay withinthe aqueous phase. Through washing, sample matrix can be removed whilecompounds of interest remain on the surface. The compounds of interestcan be removed from the surface at a later point in time by otherhigh-affinity solvents. Repeating the process helps desalt and alsoconcentrate the original sample.

In certain embodiments, chemicals are applied to the porous material tomodify the chemical properties of the porous material. For example,chemicals can be applied that allow differential retention of samplecomponents with different chemical properties. Additionally, chemicalscan be applied that minimize salt and matrix effects. In otherembodiments, acidic or basic compounds are added to the porous materialto adjust the pH of the sample upon spotting. Adjusting the pH may beparticularly useful for improved analysis of biological fluids, such asblood. Additionally, chemicals can be applied that allow for on-linechemical derivatization of selected analytes, for example to convert anon-polar compound to a salt for efficient electrospray ionization.

In certain embodiments, the chemical applied to modify the porousmaterial is an internal standard. The internal standard can beincorporated into the material and released at known rates duringsolvent flow in order to provide an internal standard for quantitativeanalysis. In other embodiments, the porous material is modified with achemical that allows for pre-separation and pre-concentration ofanalytes of interest prior to mass spectrum analysis.

In certain embodiments, the porous material is kept discrete (i.e.,separate or disconnected) from a flow of solvent, such as a continuousflow of solvent. Instead, sample is either spotted onto the porousmaterial or swabbed onto it from a surface including the sample. Adiscrete amount of extraction solvent is introduced into the port of theprobe housing to interact with the sample on the substrate and extractone or more analytes from the substrate. A voltage source is operablycoupled to the probe housing to apply voltage to the solvent includingthe extract analytes to produce ions of the analytes that aresubsequently mass analyzed. The sample is extracted from the porousmaterial/substrate without the need of a separate solvent flow.

A solvent is applied to the porous material to assist inseparation/extraction and ionization. Any solvents may be used that arecompatible with mass spectrometry analysis. In particular embodiments,favorable solvents will be those that are also used for electrosprayionization. Exemplary solvents include combinations of water, methanol,acetonitrile, and tetrahydrofuran (THF). The organic content (proportionof methanol, acetonitrile, etc. to water), the pH, and volatile salt(e.g. ammonium acetate) may be varied depending on the sample to beanalyzed. For example, basic molecules like the drug imatinib areextracted and ionized more efficiently at a lower pH. Molecules withoutan ionizable group but with a number of carbonyl groups, like sirolimus,ionize better with an ammonium salt in the solvent due to adductformation.

Discontinuous Atmospheric Pressure Interface (DAPI)

In certain embodiments, a discontinuous atmospheric pressure interface(DAPI) is used with systems and methods of the invention. Discontinuousatmospheric interfaces are described in Ouyang et al. (U.S. Pat. No.8,304,718 and PCT application number PCT/US2008/065245), the content ofeach of which is incorporated by reference herein in its entirety.

An exemplary DAPI is shown in FIG. 5. The concept of the DAPI is to openits channel during ion introduction and then close it for subsequentmass analysis during each scan. An ion transfer channel with a muchbigger flow conductance can be allowed for a DAPI than for a traditionalcontinuous API. The pressure inside the manifold temporarily increasessignificantly when the channel is opened for maximum ion introduction.All high voltages can be shut off and only low voltage RF is on fortrapping of the ions during this period. After the ion introduction, thechannel is closed and the pressure can decrease over a period of time toreach the optimal pressure for further ion manipulation or mass analysiswhen the high voltages can be is turned on and the RF can be scanned tohigh voltage for mass analysis.

A DAPI opens and shuts down the airflow in a controlled fashion. Thepressure inside the vacuum manifold increases when the API opens anddecreases when it closes. The combination of a DAPI with a trappingdevice, which can be a mass analyzer or an intermediate stage storagedevice, allows maximum introduction of an ion package into a system witha given pumping capacity.

Much larger openings can be used for the pressure constrainingcomponents in the API in the new discontinuous introduction mode. Duringthe short period when the API is opened, the ion trapping device isoperated in the trapping mode with a low RF voltage to store theincoming ions; at the same time the high voltages on other components,such as conversion dynode or electron multiplier, are shut off to avoiddamage to those device and electronics at the higher pressures. The APIcan then be closed to allow the pressure inside the manifold to dropback to the optimum value for mass analysis, at which time the ions aremass analyzed in the trap or transferred to another mass analyzer withinthe vacuum system for mass analysis. This two-pressure mode of operationenabled by operation of the API in a discontinuous fashion maximizes ionintroduction as well as optimizing conditions for the mass analysis witha given pumping capacity.

The design goal is to have largest opening while keeping the optimumvacuum pressure for the mass analyzer, which is between 10⁻³ to 10⁻¹⁰torr depending the type of mass analyzer. The larger the opening in anatmospheric pressure interface, the higher is the ion current deliveredinto the vacuum system and hence to the mass analyzer.

An exemplary embodiment of a DAPI is described herein. The DAPI includesa pinch valve that is used to open and shut off a pathway in a siliconetube connecting regions at atmospheric pressure and in vacuum. Anormally-closed pinch valve (390NC24330, ASCO Valve Inc., Florham Park,N.J.) is used to control the opening of the vacuum manifold toatmospheric pressure region. Two stainless steel capillaries areconnected to the piece of silicone plastic tubing, the open/closedstatus of which is controlled by the pinch valve. The stainless steelcapillary connecting to the atmosphere is the flow restricting element,and has an ID of 250 μm, an OD of 1.6 mm ( 1/16″) and a length of 10 cm.The stainless steel capillary on the vacuum side has an ID of 1.0 mm, anOD of 1.6 mm ( 1/16″) and a length of 5.0 cm. The plastic tubing has anID of 1/16″, an OD of ⅛″ and a length of 5.0 cm. Both stainless steelcapillaries are grounded. The pumping system of the mini 10 consists ofa two-stage diaphragm pump 1091-N84.0-8.99 (KNF Neuberger Inc., Trenton,N.J.) with pumping speed of 5 L/min (0.3 m3/hr) and a TPD011 hybridturbomolecular pump (Pfeiffer Vacuum Inc., Nashua, N.H.) with a pumpingspeed of 11 L/s.

When the pinch valve is constantly energized and the plastic tubing isconstantly open, the flow conductance is so high that the pressure invacuum manifold is above 30 torr with the diaphragm pump operating. Theion transfer efficiency was measured to be 0.2%, which is comparable toa lab-scale mass spectrometer with a continuous API. However, underthese conditions the TPD 011 turbomolecular pump cannot be turned on.When the pinch valve is de-energized, the plastic tubing is squeezedclosed and the turbo pump can then be turned on to pump the manifold toits ultimate pressure in the range of 1×10⁵ torr.

The sequence of operations for performing mass analysis using ion trapsusually includes, but is not limited to, ion introduction, ion coolingand RF scanning. After the manifold pressure is pumped down initially, ascan function is implemented to switch between open and closed modes forion introduction and mass analysis. During the ionization time, a 24 VDC is used to energize the pinch valve and the API is open. Thepotential on the rectilinear ion trap (RIT) end electrode is also set toground during this period. A minimum response time for the pinch valveis found to be 10 ms and an ionization time between 15 ms and 30 ms isused for the characterization of the discontinuous API. A cooling timebetween 250 ms to 500 ms is implemented after the API is closed to allowthe pressure to decrease and the ions to cool down via collisions withbackground air molecules. The high voltage on the electron multiplier isthen turned on and the RF voltage is scanned for mass analysis. Duringthe operation of the discontinuous API, the pressure change in themanifold can be monitored using the micro pirani vacuum gauge (MKS 925C,MKS Instruments, Inc. Wilmington, Mass.) on Mini 10.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1: Materials

Chromatography paper (grade 1) used for sample loading strip waspurchased from Whatman (Whatman International Ltd., Maidstone, ENG).Borosilicate glass tube (0.86 mm, id) modified for nanoESI tip waspurchased from Sutter Instrument (Sutter Instrument Co, Novato, Calif.,US). All the organic solvent without specified were supplied by MacronChemicals (Avantor Performance Materials Inc., Phillipsburg, N.J., US).Bovine whole blood (with EDTAK₂ as anticoagulant) was purchased fromInnovative Research (Novi, Mich., US). All other reagents were purchasedfrom Sigma-Aldrich (Milwaukee, Wis., US).

Example 2: Sample Preparation

All analytes were dissolved into methanol: H₂O 50:50 (v: v) for stocksolutions. Orange homogenate was prepared by homogenizing 10 g of orangein 10 mL of water. Porcine homogenate was prepared with 2 g of pork in15 mL of water. For imitating raw samples, analytes from stock solutionswere directly diluted to low concentrations using matrices as solvents.

Example 3: Extraction Spray

Samples used in the study were first loaded by direct pipetting 0.2 μLsample solutions onto the sample substrate, a paper strip (1 cm length,0.5 mm width, 0.18 mm thickness, grade 1), and dried in air for 1 hrbefore loading. An extraction spray source was assembled by insertingthe sample substrate to a glass nanoESI tube (0.86 mmID). Organicsolvent of 10 μL, such as MeOH and acetonitrile, was filled into thetube for analyte extraction and subsequent spray facilitated with a DCvoltage about 2 kV applied through a wire electrode (FIG. 1A).

Example 4: Mass Spectrometric Analysis

Extraction solvent and signal stability assessment were performed usinga TSQ Quantum Access Max (Thermo Scientific, San Jose, Calif.) with aheated capillary API in the product ion mode and the single reactionmonitoring (SRM) mode. The instrument settings were as followed:methamphetamine: m/z 150; collision energy: 20; scan time: 0.500 andsunitinib m/z 399→283; tube lens: 130 V; Q2 offset: 18 V.

Other assessments were completed using an AB Sciex QTRAP4000 (Sciex,Foster City, Calif.) with a curtain gas API. Typical instrumentalparameters were set as follows: spray voltage 2 kV, curtain gas, 10 psi;de-clustering potential (DP), 20 V; scan rate, 1000 Da/s.

Example 5: Mass Spectrometric Analysis with Miniature Mass Spectrometer

Limit of detection (LOD) and limit of quantitation achieved with Mini 12(L. Li, Y. Ren, T.-C. Chen, Z. Lin, R. G. Cooks and Z. Ouyang“Development and Performance Characterization of a Personal MassSpectrometry System”, 61st ASMS Conference on Mass Spectrometry andAllied Topics, Minneapolis, Minn., Jun. 9-13, 2013, MP 330) andextraction spray (FIG. 4).

LOD:

-   -   Better than 10 ng/ml for Verapamil in blood with extraction        spray

LOQ:

-   -   7.5 ng/ml Amitriptyline in blood with extraction spray (with IS)

What is claimed is:
 1. A system for analyzing a sample, the systemcomprising: an ionization probe, the probe comprising: a hollow bodythat comprises a distal tip; a paper substrate configured to hold asample, the paper substrate being at least partially disposed within thehollow body and positioned prior to the distal tip such that an analytein the sample extracted from the paper substrate by a solvent flows intothe hollow body prior to exiting the distal tip, wherein the hollow bodyis devoid of separation material after the paper substrate; and anelectrode disposed prior to the distal tip of the hollow body thatoperably interacts with the extracted analyte in the solvent to expelthe sample from the distal tip and produce ions of the analyte; and amass analyzer operably coupled to the probe to receive the ions of theprobe.
 2. The system according to claim 1, wherein the hollow body iscomposed of glass.
 3. The system according to claim 1, wherein the papersubstrate is filter paper.
 4. The system according to claim 1, whereinthe mass analyzer is for a mass spectrometer or a miniature massspectrometer.
 5. The system according to claim 4, wherein the massanalyzer is selected from the group consisting of: a quadrupole iontrap, a rectalinear ion trap, a cylindrical ion trap, a ion cyclotronresonance trap, and an orbitrap.
 6. The system according to claim 1,further comprising a source of nebulizing gas.
 7. The system accordingto claim 6, wherein the source of nebulizing gas is configured toprovide pulses of gas.
 8. The system according to claim 6, wherein thesource of nebulizing gas is configured to provide a continuous flow ofgas.
 9. A method for analyzing a sample, the method comprising:introducing a solvent to a sample held by a paper substrate that is atleast partially disposed within a hollow body comprising a distal tip,wherein the solvents interacts with the sample held by the papersubstrate to extract an analyte from the sample into the solvent,wherein the hollow body is devoid of separation material after the papersubstrate; applying a voltage to the extracted analyte in the solvent inthe hollow body from an electrode disposed prior to the distal tip ofthe hollow body to expel the sample from the distal tip of the body,thereby generating ions of the analyte; and analyzing the ions.
 10. Themethod according to claim 9, wherein a nebulizing gas is also applied tothe extracted sample.
 11. The method according to claim 10, wherein thenebulizing gas is pulsed.
 12. The method according to claim 10, whereinthe nebulizing gas is provided as a continuous flow of gas.
 13. Themethod according to claim 9, wherein the paper substrate is filterpaper.
 14. The method according to claim 9, wherein the mass analyzer isfor a mass spectrometer or a miniature mass spectrometer.
 15. The methodaccording to claim 9, wherein the sample is introduced to the papersubstrate prior to the paper substrate being inserted into the hollowbody.
 16. The method according to claim 9, wherein the sample isintroduced to the paper substrate after the paper substrate has beeninserted into the hollow body.